Protein having pesticidal activity, dna encoding the protein, and noxious organism-controlling agent and method

ABSTRACT

Noxious organism-controlling agent of the present invention is effective to pests that have acquired a resistance to conventional Bt agents and has activity on Coleoptera pests of which only several kinds have been reported.  
     Also, a novel microbe  Bacillus thuringiensis serovar galleriae  SDS 502  strain having an ability of producing a toxic protein that can serve as an active ingredient of a noxious organism-controlling agent or a protein having a pesticidal activity produced by the strain, a protein having an amino acid sequence obtainable from the amino acid sequence of the protein by addition, deletion or substitution of a plurality of amino acids and having similar pesticidal activity, a DNA encoding the protein having pesticidal activity, a microbe transformed with the DNA, a plant transformed with the DNA and its seed, as well as a noxious organism-controlling agent and method are disclosed.

DESCRIPTION

[0001] 1. Technical Field

[0002] The present invention relates to a protein having a pesticidal activity, DNA encoding the protein, a noxious organism-controlling agent and -controlling method as well as to a novel Bacillus thuringiensis serovar galleriae SDS502 strain (hereinafter, sometimes abbreviated as SDS502).

[0003] 2. Background Art

[0004]Bacillus thuringiensis (hereinafter, sometimes abbreviated as Bt) forms endospores like other Bacillus bacteria. The spores germinate and grow into vegetative cells in the presence of suitable nutritional components. The vegetative cells repeat cell division successively and sooner or later turn into sporangia that form endospores and crystal protein in the cells due to exhaustion of nutritional components, environmental changes, and so forth. Further, the cells are destructed to release the endospores and crystal protein.

[0005] Insects eat the spores and crystal protein Bt produces. When they reach the mesenteron in the digestive tract, the protein is dissolved under strongly alkaline conditions of the digestive juice to produce a protoxin, which then is converted by a proteolytic enzyme into an active ingredient (toxin). The active ingredient binds to a receptor in an epithelial cell of the mesenteron to injure cells in the vicinity of it. In the injured part, the digestive juice and the body fluid mix with each other to change the osmotic pressure and pH in the body. As a result, the food digesting function is disturbed, paralysis of mouth-part is caused, and the feeding action is retarded in the insect. Furthermore, the spores germinate under nutritional conditions and they invade into the hemocele of the insect according as the vegetative cells propagate, thus causing blood poisoning.

[0006] Although insects may have different sensitivity depending on the species of insect, usually the feeding action ceases after several hours and the insect dies after 2 or 3 days after eating Bt. It is attributable to this phenomenon that less damage by insects' eating is observed even when some insects remain alive after Bt is used. Many synthetic insecticides act on the nerve system of insect so that vigorous convulsion or knockdown effect, paralysis or the like phenomenon is observed. However, the mechanism of the action of Bt is quite different as described above and the effect is gradually exhibited even though living insects exist after the treatment. Bt and protein having a pesticidal activity (crystalline toxic protein) produced by Bt are very useful as an environmentally safe microbial pesticides (Bt agents), in particular as insecticides for Lepidoptera insects and are practically used worldwide.

[0007] Bt is gram-positive rod cells and produces crystal protein in the spore formation stage at a late stage of logarithmic phase. The crystal protein is not converted into a protein having a pesticidal activity to cause gut paralysis and systemic paralysis before it is orally taken into the digestive tract by an insect to be subjected to alkali decomposition and enzymatic decomposition in the digestive juice. However, it does not exhibit toxicity to mammals.

[0008] The crystal proteins Bt produces are formed in the sporangium along with the spores and released to the outside of the cell together with the spores after passing the phase of the sporangium (Nature, 172, 1004, 1953). These generally constitute complex crystals such as diamond-shaped, bipyramidal, rhomboidal and so forth and are insoluble in water. They are produced one per spore at the time of spore formation and released together with spores into medium according as the bacterial cell is destructed. Usually they are of a steric rhombic or orthorhombic structure and have a size on the order of 2.0μ in the long side and 0.6μ in the short side. Subspecies include also amorphous ones and their size varies widely. On their surface, regular stripe structures can be seen. Isolation from the medium and purification of crystal protein can be performed by use of a bilayer fractionation method, a density gradient centrifugation method or the like.

[0009] The crystal proteins are soluble in an NaOH solution having a pH 12 or more. According to the analyses by SDS-PAGE (polyacrylamide gel electrophoresis), there are observed three proteins of about 130 to 135 kDa, about 65 kDa and about 80 kDa in a bacterial strain belonging to Bacillus thuringiensis. They are generically called Cry 1 protein, Cry 2 protein, and Cry 5 protein. Furthermore, they can be separated into a plurality of proteins that have almost approximate molecular weights but partially differ from each other by a fractionation operation such as high performance liquid chromatography. That is, in the case of Cry-1 protein, it is classified into proteins Cry-1Aa, Cry1Ab and so forth.

[0010] Bt was isolated from larva of Mediterranean flour moth (Ephestia kuehniella Zeller [Pyralidae]) by Berliner, a German researcher in 1911. Since the larva of the insect ate the flour from Thuringia, the insect was named Thuringiensis. Earlier than this, Dr. Ishiwatari isolated the same bacterial species as a pathogenic bacterium to silkworm in 1901. Thus, it is understood that Bt has widely occurred in the natural world since old. For example, it is present in grain warehouses and millhouses where grain pests inhabit. Also, it is detected in wagons and cabins and so forth for transporting grains. Thus it is known that it migrates everywhere in the world. Also in Japan, its distribution in every district has been examined and many Bacillus thuringiensis strains have been isolated from the dust in the houses of silkworm farmers, the surface of plants and so forth.

[0011] The bacteria that belong to the genus Bacillus amount to 70 or more species. Those strains frequently observed worldwide include 22 strains. They are distinguished basically by the ability of spore formation and shape of spore, production of gas, production of acetylmethylcarbinol (AMC), reduction of nitrates, and assimilability of some sugars in accordance with the techniques of Thiery and Franchon. Bacillus thuringiensis (B. thuringiensis) is finally distinguished from its allied species by the presence or absence of a crystal having a pesticidal activity (“Manual of techniques in insect pathology,” L. Lacey ed., Academic Press, California, 55-77 (1997)).

[0012] The characteristics used for distinguishing Bacillus thuringiensis from other bacterial species and other species belonging to the genus Bacillus include gram-positive rod, catalase (+), spore formation (+), ovary spore, 0.9μ or more in width of vegetative cell, production of acetylmethylcarbinol (+), facultative anaerobicity, assimilation of D-mannitol (−), and existence of crystal protein (+).

[0013] For the identification of subspecies of Bt, flagellum antigen (H-antigen) according to the serological technique by De Barjac and Bonefoi using an antibody in a rabbit serum to the flagellum of a bacterium has been employed for a long time as long as 40 years (Entomophaga 7, 5-31, 1962). This is a technique that has been widely utilized for the phylogenetic systematics of Bacillus thuringiensis.

[0014] The pesticidal activity of the bacterial strains varies depending on subspecies and is of an extremely high specificity. For example, there have been known kurustaki, aizawai and so forth as subspecies that exhibit an activity to Lepidoptera insects and tenebrionis, japonensis and so forth as subspecies that exhibit an activity to Coleoptera insects.

[0015] However, in actuality, bacterial strains belonging to the same subspecies may differ in the spectrum of pesticidal activity depending on the strain. In the case of Bt strains that have an activity to a part of Lepidoptera insect pests, the pests have acquired a resistance thereto. In addition, few reports have been made on strains exhibiting effective activity to Coleoptera insects.

[0016] Thus, a novel Bt agent that is effective to Lepidoptera insect pests having acquired a resistance to the Bt agent is demanded. Furthermore, there is a keen demand for a Bt agent having an activity to Coleoptera insects. Among these, novel Bt agents having a pesticidal activity to larvae of Coleoptera insects, in particular larvae of scarabs, thus far reported include only Bacillus thuringiensis Serovar. japonensis strain buibui) strain (Japanese Patent Application Laid-open Nos. Hei 6-65292 and Hei 7-179) and Bacillus thuringiensis var. japonensis N141 (Japanese Patent Application Laid-open No. Hei 8-228783).

DISCLOSURE OF THE INVENTION

[0017] The conventional buibui strain or N141 strain belonging to the subspecies japonensis does not exhibit sufficient effect on larvae of scarabs, in particular larvae of Anomala cuprea, a serious pest for lawn grasses, taro, sweet potato, peanut and so forth, and Anomala orientalis and Popillia japonica, pests for lawn grasses. Furthermore, Bt toxins of the microbe that belongs to the same bacterial species (subspecies) exhibit crossing in case a resistance is developed in a part thereof, with the result that its effect is considerably decreased. On the other hand, Bt toxins take a certain time for their effect to be exhibited. Therefore, discovery of a novel toxin that has a more potent pesticidal activity is keenly desired.

[0018] Therefore, an object of the present invention is to provide a novel bacterial strain belonging to Bacillus thuringiensis serovar galleriae that produces a pesticidal protein having a high pesticidal activity to larvae of Coleoptera insects and to provide a protein having a pesticidal activity derived from the novel microbe.

[0019] Furthermore, an object of the present invention is to provide a protein that has the above pesticidal activity, a protein that has an amino acid sequence obtained by addition, deletion or substitution of a plurality of amino acids in the amino acid sequence that constitutes the protein and has the same pesticidal activity, DNAs that encode such amino acid sequences, microbes that have been transformed by use of the DNAs and produce proteins having pesticidal activity, plants transformed by use of such DNAs or seeds thereof, and noxious organism-controlling agents and -controlling methods.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 is an electron micrograph of Bacillus thuringiensis serovar galleriae SD502 strain.

[0021]FIG. 2 is a diagram illustrating the results of SDS-PAGE of crystal protein having a pesticidal activity of the present invention. 1 designates markers, showing 200, 116.25, 97.4, 66.2, and 45.0 kDa from the top. 2 represents the results of crySDS502 gene product expressed in Escherichia coli. 3 represents the results of SDS 502 crystal protein.

[0022]FIG. 3 is a diagram illustrating linkage of Bacillus thuringiensis serovar galleriae SD502 gene to a vector (gene cassette).

DETAILED DESCRIPTION OF THE INVENTION

[0023] With a view to finding out a novel microbe having a high effect on larvae of Coleoptera insects, the present inventors have conducted repeated analyses and as a result they have found out a Bacillus thuringiensis serovar galleriae SD502 strain belonging to Bacillus thuringiensis serovar galleriae that produces a pesticidal protein having a high pesticidal activity to larvae of Coleoptera insects. Thus, they have achieved the present invention that relates to a pesticide containing the novel Bacillus thuringiensis serovar galleriae SD502 itself and/or a pesticidal protein (toxic protein) it produces as active ingredient(s).

[0024] Furthermore, they have confirmed that a DNA encoding the pesticidal protein the novel microbe of the present invention produces, a protein having an amino acid sequence encoded by the DNA, and a noxious organism-controlling agent containing the protein as an active ingredient are effective as pest-controlling means and thus achieved the present invention.

[0025] That is, the present invention relates to (1) proteins having a pesticidal activity, (2) DNA encoding such proteins, (3) noxious organism-controlling agents, (4) plant protectingmethods, (5) (5-1) microbes, (5-2) plants or seeds thereof transformed by use of the DNA, and (6) novel microbe, as set forth below.

[0026] 1) A protein having an amino acid sequence described in SEQ. ID. No. 1 in the Sequence Listing and exhibiting a pesticidal activity.

[0027] 2) A protein having an amino acid sequence derived by addition, deletion or substitution of a plurality of amino acids in the amino acid sequence described in SEQ. ID. No. 1 in the Sequence Listing and exhibiting a pesticidal activity.

[0028] 3) A DNA containing a nucleotide sequence encoding the protein as described in 1) above.

[0029] 4) The DNA as described in 3) above, containing a nucleotide sequence as described in SEQ. ID. No. 3 in the Sequence Listing.

[0030] 5) A DNA containing a nucleotide sequence encoding the protein as described in 2) above.

[0031] 6) A noxious organism-controlling agent, comprising

[0032] a microbe producing a protein having an amino acid sequence described in SEQ. ID. No. 1 in the Sequence Listing, selected from

[0033] (1-1) Bacillus thuringiensis serovar galleriae SD502 strain,

[0034] (1-2) a mutant thereof, and

[0035] (1-3) a microbe transformed with a DNA containing a nucleotide sequence encoding a protein having an amino acid sequence described in SEQ. ID. No. 1 in the Sequence Listing, or

[0036] a protein having a pesticidal activity, produced by a microbe selected from

[0037] (2-1) the above-mentioned SDS502 strain,

[0038] (2-2) its mutant, and

[0039] (2-3) transformed microbe.

[0040] 7) A microbe transformed with the DNA as described in 5) above and producing a protein exhibiting a pesticidal activity as described in 2) above.

[0041] 8) A plant transformed with the DNA as described in 3) or 5) above, or seed thereof.

[0042] 9) A method for controlling a noxious organism, wherein the protein as described in 1) or 2) above is fed to a noxious organism to protect a plant from a damage caused by the noxious organism.

[0043] 10) The method for controlling a noxious organism as described in 9) above, wherein the noxious organism is a Coleoptera insect and the plant is protected from a damage caused by the noxious organism.

[0044] 11) Bacillus thurinqiensis galleriae SDS502strain producing a protein having an amino acid sequence described in SEQ. ID. No. 1 in the Sequence Listing and exhibiting a pesticidal activity.

[0045] Novel Bacillus thuringiensis serovar galleriae SDS502 strain of the present invention has been internationally deposited at National Institute of Advanced Industrial Science and Technology, Independent Administrative Institution, under Accession No. FERM BP-7667.

[0046] The SDS502 strain can be cultured in a medium in which general bacteria can grow by a common fermentation technique.

[0047] Examples of medium include a common broth medium (0.3% of meat extract, 1.0% of peptone, and 0.5% of NaCl, pH 7.0), an MBS medium (0.7% of KH₂PO₄, 1% of bactotryptose, 0.2% of yeast extract, 0.03% of MgSO₄.7H₂O, and 0.02% of CaCl₂.2H₂O, pH 7.2), an MRVP medium (0.5% of polypeptone, 0.5% of glucose, and 0.5% of NaCl, pH 7.0) and so forth.

[0048] As the carbon source, glucose, fructose, saccharose, maltose, molasses, soluble starch, cornstarch and so forth may be utilized.

[0049] As the nitrogen source, ammonium chloride, ammonium sulfate, urea, yeast extract, peptone, soybean powder, casein and so forth may be utilized.

[0050] Furthermore, it is preferable that as other inorganic salts and vitamins, NaH₂PO₄, K₂HPO₄, MnSO₄, FeSO₄, MgSO₄, NaCl, molasses, yeast extract, EBIOS (vitamin preparation) and so forth be added. The pH is preferably 6 to 8. The incubation temperature is preferably 25 to 33° C. The incubation time is preferably 24 to 120 hours. The culture method is preferably the one under aerobic conditions, such as aerobic spinner culture.

[0051] In the case where pesticidal crystal protein is isolated from the culture broth after the incubation, a common centrifugal separation method, a filtration method and so forth may be utilized. Alternatively, the SDS502 strain and/or crystal protein the SDS502 strain produces may be used in the form of a mixture with vegetative cells and/or spores without separation therefrom.

[0052] Also, mutant strains that produce a pesticidal crystal protein may be obtained from the SDS502 strain as an original strain by spontaneous or induced mutation, which strains may be used as an insecticidal crystal protein-producing strain according to the present invention. As the method for making mutant strains, a common method conventionally known can be used, for example, a method in which an original strain is subjected to artificial mutation by irradiation of ultraviolet rays or with a chemical such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG), spread on an agar medium containing skimmed milk, screening a colony forming a greater clear zone around the colony from among the strains that grow thereon, and screening a strain having an excellent productivity.

[0053] In the case where a noxious organism-controlling agent containing the SDS502 strain and/or SDS502 crystal protein as an active ingredient or ingredients, it may be made into an optional formulation such as wettable powder, granules, dust, flowable formulation in the same manner as in common pesticides. These are used in admixture with a suitable carrier for respective formulations, for example, powder of a mineral such as agalmatolite, talc, kaolin, calcium carbonate, bentonite, silica stone powder, limestone powder, acid clay, diatomaceous earth powder, gypsum, pumice powder, shell powder, mica powder, or colloidal hydrated sodium silicate, water, or aqueous solutions such as buffer solutions. Preferably, they are used after addition of a fixing agent such as an alkylbenzenesulfonate or an alkylsulfonate, a humectant such as a polyoxyethylene (POE) alkyl ether, a POE alkyl phenyl ether, a POE dialkyl phenyl ether, a POE alkylamine or a dialkyl sulfosuccinate, a dispersing agent such as an alkyl sulfate, a POE alkyl ether sulfate, a POE alkyl phenyl ether sulfate, a POE benzylated (or salicylated) phenyl (or phenylphenyl) ether sulfate, a paraffin (alkane) sulfonate, an alpha-olefin sulfonate (AOS), an alkyl benzenesulfonate, a mono- or dialkyl naphthalene sulfonate, a naphthalenesulfonate/formaldehyde condensate, an alkyl diphenyl ether disulfonate, a lignin sulfonate, a POE alkyl ether sufosuccinate half ester, or a POE benzyl (or styrylated) phenyl (or phenylphenyl) ether phosphate, a mildewproofing agent such as a paraoxybenzoic acid derivative, salicyl anilide, 1,2-benzoisothiazolin-3-one, tetraphthalonitrile (TPN) or 2-nitrobromo compound.

[0054] In contrast to using the SDS502 strain and/or SDS502 strain-produced crystal protein as a single active ingredient, it is also possible to mix it with herbicides, various pesticides, bactericides or plant growth regulators which are effective to other noxious organisms, synergists for multiplying the effect, attractants as well as plant nutritive agents, fertilizers and so forth that are intended to obtain other functions.

[0055] In preparing a noxious organism-controlling agent containing the SDS502 strain and/or SDS502 strain-produced crystal protein as an active ingredient or ingredients, its active ingredient content is suitably on the order of 10 to 99%, preferably 40 to 90%. However, the active ingredient content may be adjusted depending on the target noxious organism, cultivated crop, method of use, time of use and so forth.

[0056] The crystal protein of the present invention includes, in addition to those having the amino acid sequence as described in SEQ. ID. No. 1 in the Sequence Listing, also those having the one that is partly deficient (for example, a polypeptide composed of only a portion that is necessary for the expression of bioactivity out of the amino acid sequence as described in SEQ. ID. No. 1 in the Sequence Listing), those having the one partly substituted with other amino acids (for example, the one substituted by amino acids having similar physical properties), and those in which other amino acids are added or inserted in some part thereof.

[0057] As is well known in the art, 1 to 6 kinds of codon are known to code for one amino acid (for example, one kind for Met and 6 kinds for Leu). Therefore, The nucleotide sequence of DNA can be altered without altering the amino acid sequence of a polypeptide.

[0058] Examples of the pests that can be controlled by the method of the present invention include the following Coleoptera insects. That is, scarabs such as Anomala cuprea, Anomala diversa, Anomala octiescostata, Hoplia communis, Ectinohoplia obducta, Anomala orientalis, Anomala osakana, Anomala testaceipes, Anomala schonfeldti, Anomala rufocuprea, Anomala albopilosa, Maladera castanea, Melolontha japonica, Adoretus tenuimaculatus and Popillia japonica, ladybugs such as Epilachna vigintioctopunctata and Epilachna vigintioctomaculata, weevils such as Lissorhoptrus oryzophilus, Scepticus griseus, Cylas formicarius, Sphenophrus venatus vestius and Sitophilus zeamaise, leaf beetles such as Phyllotreta striolata and Aulacophora femoralis, click beetle such as Melanotus okinawaensis, long-horned beetles such as Monochamus alternatus and Mesosa myops, bark beetles such as Scolytus japonicus and Xylosandrus germanus, and confused flour beetles such as Tenebrio molitor and Tribolium castaneum.

[0059] The method for controlling a noxious organism of the present invention using a noxious organism-controlling agent that contains the SDS502 strain and/or SDS502 crystal protein as an active ingredient or ingredients can be used for protecting a wide variety of plants that are susceptible to attack of Coleoptera insect pests. Specific examples of target plant include vegetables such as Chinese cabbage and cabbage, fruit vegetables such as cauliflower, root crop such as sweet potato or taro, citrus, defoliating fruit trees, cereals such as rice, wheat and beans, lawn grass in golf courses, gardens and so forth, specialty crop such as tea or sugarcane, stored cereals, stored food and flower trees. Also, the method of the present invention can be used for trees in forestation and non-agricultural areas such as parks, trees in forests, and seedling and so forth.

[0060] Generally, the method for protecting plants from insect damages by Coleoptera insect pests by use of a noxious organism-controlling agent that contains the SDS502 strain and/or SDS502 crystal protein as an active ingredient or ingredients can be practiced by treating (for example, spraying on) a plant where insect pests proliferate or will tend to proliferate with a composition of the above-mentioned noxious organism-controlling agent diluted with a diluent such as water, or directly mixing or injecting into soil without dilution.

[0061] The SDS502 gene can be isolated from the SDS502 strain. Total DNA of the SDS502 strain is digested with one or more restriction enzymes and the produced DNA fragment is converted to a 2- to 5-kbp DNA fraction. The fraction is linked to a suitable vector and Escherichia coli is transformed therewith. Next, using an antibody to the pesticidal crystal protein the SDS502 strain produces, an enzyme immunoassay method is practiced and thus an Escherichia coli transformant having the objective gene can be obtained.

[0062] The crystal protein gene DNA derived from the SDS502 strain thus obtained is treated with a suitable restriction enzyme or enzymes and the obtained DNA fragment is coupled to a suitable cloning vector to make a gene cassette. Using this, microbes such as Escherichia coli and Bacillus subtilis can be transformed. For example, Escherichia coli can be transformed and a nucleotide sequence encoding the SDS502 strain produced crystal protein can be analyzed by a gene analysis method such as a dideoxy method.

[0063] Using the gene cassette, gram-positive bacteria having a pesticidal activity, for example, Bacillus thuringiensis serovar galleriae or other subspecies can be transformed. This enables one to obtain transformed Bacillus thuringiensis effective for controlling a broader range of insects.

[0064] Furthermore, to express the SDS502 gene in plants, a preferred restriction site may be introduced so as to be located on a flank of each gene or gene part to induce mutation of a specified site.

[0065] The SDS502 gene part encoding the active part of the pesticidal crystal protein of the SDS502 strain can be stably inserted in the nuclear genome in a single plant cell to make a transformed plant having a resistance to insects or having the ability of killing insects.

[0066] As a result, using the obtained transformed plant, transformed plants having the same characteristics can be produced. Furthermore, the SDS502 gene part having a resistance to insects or the ability of killing insects can be introduced into other variants of the same or related plant species. The seeds obtained from the transformed plants are stable genome inserted products that contain the SDS502 gene part that can exhibit a resistance to insects or insecticidal activity and that is effective as a pesticide.

[0067] The SDS502 strain may be further transformed with one or more exogenous Bt genes having pesticidal activities. For example, the noxious organisms on which the SDS502 strain and/or SDS502 strain-produced crystal protein has no activity includes in particular larvae of Lepidoptera insects. Now, a chimera gene of the SDS502 gene with a gene encoding a crystal protein derived from other microbe exhibiting an effective activity on them may be prepared and used for transforming the microbe to the one having a wider pesticidal spectrum. By so doing, transformed SDS502 strains that can control a wider variety of pests are produced.

[0068] Antibody specific to SDS502 strain crystal protein can be prepared by immunizing a guinea pig with the crystal protein of the SDS502.

BEST MODE FOR CARRYING OUT THE INVENTION

[0069] Hereinafter, the present invention will be illustrated by way of Examples. However, the present invention is by no means limited by the following Examples.

EXAMPLE 1

[0070] Isolation of Bacillus thuringiensis serovar galleriae SDS502 Strain

[0071] From soil collected in the city of Tsukuba, a Bacillus thuringiensis serovar galleriae SDS502 strain was isolated by use of the following technique.

[0072] 10 mg of sample soil was charged in an Erlenmeyer flask and 10 ml of sterilized water was poured therein. After shaking for 30 minutes, the mixture was left to stand for a while. Then, 2 ml of supernatant was taken out and immediately heated at 80° C. for 10 minutes. The heated liquid was diluted in two stages to 10 folds and further to 100 folds. 1 ml each of dilutions was incubated on an NB plate medium (0.3% of meat extract, 1.0% of peptone, 10.5% of NaCl, 2% of agar, pH 7.0/distilled water) at 30° C. for 24 to 48 hours.

[0073] Out of the obtained colonies, white, rough-edged and rapidly growing colonies were selected to obtain Bacillus thuringiensis in a high probability.

EXAMPLE 2

[0074] Bacteriological Properties of Bacillus thuringiensis serovar galleriae SDS502 Strain Method: Search was conducted in accordance with the taxonomy and bacteriological techniques described in Cowan. S. T., “Manual of Identification of Medical Bacteria” (translated by T. Sakazaki, Kindai Shuppan).

[0075] Gram stain: Gram-positive rod,

[0076] Morphology of colony: Forms an opaque beige colony having irregular edges,

[0077] Spore forming ability and shape of spore: (+) oval spore;

[0078] Catalase: (+),

[0079] Width of vegetative cell: 0.9μ or more,

[0080] Production of AMC: (+),

[0081] Respiration: Facultative an aerobic,

[0082] Assimilation of D-Mannitol: (−),

[0083] Existence of crystal protein: (+),

[0084] Serum type of flagella: H anti-serum type (5a5b),

[0085] Cell contents: Spore forming cells produce amorphous type crystal protein (cf. FIG. 1),

[0086] Alkali-soluble protein: (+) protein electrophoresed near 130 kDa (cf. FIG. 2),

[0087] Activity: The strain of the invention has lethal activity on Coleoptera pests tested.

[0088] From the above findings, the strain of the invention was judged to be a novel strain. This was named Bacillus thuringiensis serovar galleriae SDS502 and deposited at Laboratory of Microbial Industry and Technology, Institute of Industrial Science Technology, Ministry of International Trade and Industry, (now National Institute of Advanced Industrial Science and Technology, Independent Administrative Institution) under Accession No. FERM P-17979 and transferred to International Deposition under International Receipt No. FERM BP-7667.

EXAMPLE 3

[0089] Identification of Subtype of Bacillus thuringiensis serovar galleriae SDS502 strain

[0090] By use of an antibody to a protein of the flagellum of a serotyping Bacillus prepared with an antibody derived from an antigen of flagellum, an antigen-antibody reaction was carried out using a flagellum protein of an unknown bacterium as an antigen.

[0091] Flagellum H serum was prepared by heating the bacteria cells at 100° C. to peel flagella off. Using 40 kinds (subtypes) of H antigen standard strains of Bacillus thuringiensis that are already known, bacteria having good mobility were selected using a Craigie tube (0.5% semi-fluid agar medium) and formalized dead bacteria were prepared from them. The formalized dead bacteria were given to a rabbit to immunize it. H serum was prepared by absorbing a corresponding antibody to the Bacillus thuringiensis cell antigen from each antiserum. The serum type of H antigen and agglutinin value of the antibody were identified and quantitatively determined according to the method of Ooba and Ayusawa (I. Invertebr. Pathol., 32, 303-309, 1978).

[0092] The H antigen to Bacillus thuringiensis serovar galleriae SDS502 strain specifically agglutinates serovar galleriae only. The agglutinin value of serovar galleriae SDS502 strain H antiserum to a corresponding homo antigen was 12,800 folds and the agglutinin value of it to serovar galleriae HD8 strain (standard strain) was 6,400 folds. Therefore, SDS502 strain and serovar galleriae were judged to be the same strain.

EXAMPLE 4

[0093] Purification of Crystal Protein of SDS502 Strain and Properties Thereof

[0094] One platinum loop of SDS502 strain cells were taken out and inoculated in a test tube containing common bouillon medium (0.3% of meat extract, 1.0% of peptone, 0.5% of NaCl, pH 7.0/distilled water). Reciprocating shaking culture of it was performed at 30° C. for 24 hours to obtain a seed culture solution. The seed culture was inoculated in a 500 ml Erlenmeyer flask containing 100 ml of the above-mentioned medium such that the seed culture was in a final concentration of 1% and rotation shaking culture was performed at 30° C. for 96 hours at 250 rpm. Then, cells, spores and crystal protein were recovered by centrifugation. A suitable amount of buffer (Tris-HCl, NaCl, EDTA) was added to the obtained precipitate and supersonic destruction was performed to obtain a suspension. The obtained suspension was subjected to 8% SDS-PAGE gel electrophoresis to examine its electrophoretic pattern. Also, using an antibody, Western blotting was performed. As result, it was confirmed that there existed a crystal protein having molecular weight of about 130 kDa produced by the SDS502 strain.

EXAMPLE 5

[0095] Pesticidal Activity of SDS502 Strain on Anomala cuprea, Popillia japonica, Anomala orientalis, Plutella xylostella, and Bombyx mori

[0096] The suspension prepared in Example 4 was diluted to a crystal protein concentration of 10 μg/ml and a spreading agent was added thereto to obtain a sample solution. The sample solution was mixed with leaf mold that had been subjected to sterilization treatment in advance and 1st stage, 2nd stage and 3rd stage larvae of Anomala cuprea, 1st stage and 2nd stage larvae of Popillia japonica, as well as 1st stage and 2nd stage larvae of Anomala orientalis were released.

[0097] Furthermore, the leaf of cabbage was dipped in the sample solution and thereafter it was sufficiently air-dried. This was placed in a Styrol cup containing wet filter paper. In the cup, larvae of Plutella xylostella in the middle phase of 3rd stage were released. After 7 days (after 5 days in the case of Bombyx mori, or after 2 days in the case of Plutella xylostella), the mortality of larvae was obtained according to the following formula. The tests were performed in 5 series with 5 insects per lot.

Mortality (%)=(Number of dead insect/Number of released insect)×100

[0098] Furthermore, the sample solution mixed with 5 g of artificial feed stuff was charged in a dish. In the dish, larvae of Bombyx mori on the 2nd day in the 3rd stage were released and the mortality of larvae after 7 days (after 5 days in the case of Bombyx mori, or after 2 days in the case of Plutella xylostella) was obtained according to the above-mentioned formula. The tests were performed in 5 series with 5 insects per lot. As a control, a test solution of the pesticidal protein produced by Bacillus thuringiensis serovar galleriae HD8 strain (standard strain) was prepared in the same manner as above and comparison therewith was made.

[0099] As a result, as indicated in the pesticidal spectrum of the crystal protein produced by Bacillus thuringiensis serovar galleriae SDS502 strain (Table 1), the pesticidal protein produced by the SDS502 strain exhibited pesticidal effect to Anomala cuprea Hope, Anomala orientalis, and Popillia japonica belonging to Coleoptera in a concentration of 10 μg/ml while the crystal protein produced by Bacillus thuringiensis serovar galleriae HD8 strain (standard strain) exhibited no pesticidal effect. On the other hand, the HD8 strain (standard strain) exhibited high activity to larvae of Bombyx mori, Plutella xylostella, and Spodoptera litura belonging to Lepidoptera while the SDS502 strain exhibited no activity to Lepidoptera insects except for Plutella xylostella. These results suggest that the crystal proteins have different compositions and the strains cannot be said to be completely the same strain in consideration of the facts that the galleriae standard strain has cry1Ab gene and exhibits a pesticidal effect to Coleoptera while the SDS502 strain exhibits substantially no pesticidal activity to Lepidoptera. TABLE 1 Mortality (%) After 7 Days From Eating Crystal Protein (10 μg) Bacillus thuringiensis serovar galleriae SDS 502 HD8 Strain Name of Insect Strain (Standard Strain) Anomala cuprea larvae 100 0 (1st stage Larvae) Anomala cuprea larvae 100 0 (2nd stage Larvae) Anomala cuprea larvae 80 0 (3rd stage Larvae) Popillia japonica 100 0 (1st stage Larvae) Popillia japonica 100 0 (2nd stage Larvae) Anomala orientalis larvae 100 0 (1st stage Larvae) Anomala orientalis larvae 100 0 (2nd stage Larvae) Bombyx mori * 0 80 Plutella xylostella. ** 40 80 Spodoptera litura 0 40

EXAMPLE 6

[0100] Gene Relating to Pesticidal Protein of Bacillus thuringiensis serovar galleriae SDS502 Strain

[0101] An antibody obtained by immunizing a guinea pig with about 130 kDa crystal protein produced by Bacillus thuringiensis serovar galleriae SDS502 strain was used for cloning a gene encoding SDS502 strain crystal protein (hereinafter, abbreviated as SDS502 gene). The cloned gene had 3,690 nucleotides and contained a translation region ranging from 187th ATG codon to 3,688th TAA codon. Further, it was compared with known genes, i.e., japonensis buibui gene (Japanese Patent Application Laid-open No. H6-65292) and japonensis N141 gene (Japanese Patent Application Laid-open No. H8-228783) effective to Coleoptera insects. As a result, it revealed that the both genes showed only 71% and 42% homologies, respectively, in amino acid sequence.

EXAMPLE 7

[0102] Isolation and Cloning of SDS502 Gene

[0103] Total DNA was prepared from SDS502 strain and partially cleaved with restriction enzyme EcoRI. From the cleaved DNA, about 2 to 5 kbp DNA fragment was obtained by fractionation and ligated with a phage vector (λgt11) cleaved with EcoRI. Escherichia coli was transformed with the product. Then, the recombinant Escherichia coli clone was subjected to antibody screening with an antibody obtained by immunizing a guinea pig with the about 130-kDa protein that was considered to be SDS502 stain crystal protein to identify a clone containing the SDS502 gene. DNA was prepared from the recombinant Escherichia coli clone and the DNA was cleaved with restriction enzyme EcoRI. The cleaved DNA fragments were electrophoresed on 0.8% agarose gel to identify an about 3.4-kbp insert DNA fragment.

[0104] The obtained DNA fragment was fractionated and ligated to Bluescript II SK(−), which was a plasmid vector cleaved with EcoRI, to prepare a gene cassette (pSDS502) (FIG. 3). Since this pSDS502 was not of full length, cloning was performed again to obtain a full-length pSDS502. Thereafter, the nucleotide sequence of the DNA fragment containing full-length SDS502 gene was determined by dideoxy method.

EXAMPLE 8

[0105] Expression SDS502 Crystal Protein in Escherichia coli (E. coli: DH5α) and Properties of Expressed Protein

[0106] To produce crystal protein using the SDS502 gene, Escherichia coli (E. coli: DH5α) was transformed by use of the gene cassette (pSDS502) to obtain a recombinant Escherichia coli (hereinafter referred to as E. coli: DH5α(pSDS502)). The recombinant Escherichia coli was incubated in LB-amp liquid medium (10 g of Trypton, 10 g of NaCl, 5 g of yeast extract, 0.2% of glucose, 50 mg/l of sterilized water of ampicillin) at 37° C. for about 3 hours. Then, isopropyl 1-thio-β-D-galactoside (IPTG) was added thereto to a final concentration of 1 mM and the cultivation was continued for additional 20 hours at 37° C. After completion of the cultivation, the culture was centrifuged. Lysisbuffer was added to the precipitate in an amount of 4 folds (W/V) and the mixture was suspended at room temperature for 10 minutes. Then Lysozyme was added thereto to a final concentration of 1 mg/ml and after mixing, the mixture was left to stand on ice for 10 minutes. Further, Triton X-100 was added to this to a final concentration of 1% and after mixing, the mixture was centrifuged and the supernatant portion thereof was recovered. The obtained supernatant was subjected to 8% SDS-PAGE gel electrophoresis to examine its electrophoretic pattern. Also, Western blotting by use of an antibody was performed. As a result, it was observed that E coli: DH5α(pSDS502) produced a crySDS502 crystal protein.

EXAMPLE 9

[0107] Pesticidal Activity of Crystal Protein Derived from E. coli: DH5α(pSDS502) on 1st stage larvae of Anomala cuprea and of Popillia japonica

[0108] The supernatant solution obtained as described above was diluted such that the concentration of crystal protein was 10 μg/ml and a spreading agent was added thereto to make a sample solution. The sample solution was mixed with leaf mold that had been subjected to sterilization treatment in advance and 1st stage larvae of Anomala cuprea and of Popillia japonica, were released. As a result, it was confirmed that the sample solution exhibited pesticidal activity on Anomala cuprea and Popillia japonica.

INDUSTRIAL APPLICABILITY

[0109] According to the present invention, a novel microbe Bacillus thuringiensis serovar galleriae SDS502 strain having ability of producing a toxic protein having high pesticidal activity on Coleoptera larvae has been found out and also a gene encoding the pesticidal crystal protein and pesticidal crystal protein have been found out. Furthermore, by formulating a noxious organism-controlling agent comprising the protein as an active ingredient, a noxious organism-controlling agent having activity on noxious organisms that have acquired a resistance to the conventional Bt can be provided. In particular, the noxious organism-controlling agent of the present invention is superior in effect on larvae of Anomala cuprea, which is a strong pest for lawn grasses, taro, sweet potato, peanut and so forth, and on Anomala orientalis and Popillia japonica and so forth, which are pests for lawn grasses, with better cost performance, as compared with the conventional pesticides produced by chemical synthesis and the buibui strain belonging to the subgenus japonensis.

1 3 1 1167 PRT Bacillus thuringiensis 1 Met Ser Pro Asn Asn Gln Asn Glu Tyr Glu Ile Leu Asp Ala Ser Ser 1 5 10 15 Ser Thr Ser Val Ser Asp Asn Ser Val Arg Tyr Pro Leu Ala Asn Asp 20 25 30 Gln Thr Thr Thr Leu Gln Asn Met Asn Tyr Lys Asp Tyr Leu Arg Met 35 40 45 Ser Glu Gly Glu Asn Pro Glu Leu Phe Gly Asn Pro Glu Thr Phe Ile 50 55 60 Ser Ser Ser Thr Val Gln Thr Gly Ile Gly Ile Val Gly Gln Val Leu 65 70 75 80 Gly Ala Leu Gly Val Pro Phe Ala Gly Gln Ile Ala Ser Phe Tyr Ser 85 90 95 Phe Ile Val Gly Gln Leu Trp Pro Ser Ser Thr Val Ser Val Trp Glu 100 105 110 Met Ile Met Lys Gln Val Glu Asp Leu Ile Asp Gln Lys Ile Thr Asp 115 120 125 Ser Val Arg Lys Thr Ala Leu Ala Gly Leu Gln Gly Leu Gly Asp Gly 130 135 140 Leu Asp Val Tyr Gln Lys Ser Leu Lys Asn Trp Leu Glu Asn Arg Asn 145 150 155 160 Asp Thr Arg Ala Arg Ser Val Val Val Thr Gln Tyr Ile Ala Leu Glu 165 170 175 Leu Asp Phe Val Ala Lys Ile Pro Ser Phe Ala Ile Ser Gly Gln Glu 180 185 190 Val Pro Leu Leu Ser Val Tyr Ala Gln Ala Ala Asn Leu His Leu Leu 195 200 205 Leu Leu Arg Asp Ala Ser Ile Phe Gly Ala Glu Trp Gly Phe Thr Pro 210 215 220 Gly Glu Ile Ser Thr Phe Tyr Asp Arg Gln Val Thr Arg Thr Ala Gln 225 230 235 240 Tyr Ser Asp Tyr Cys Val Lys Trp Tyr Asn Thr Gly Leu Asp Lys Leu 245 250 255 Lys Gly Thr Asn Ala Ala Ser Trp Leu Lys Tyr His Gln Phe Arg Arg 260 265 270 Glu Met Thr Leu Leu Val Leu Asp Leu Val Ala Leu Phe Pro Asn Tyr 275 280 285 Asp Thr Arg Thr Tyr Pro Ile Glu Thr Thr Ala Gln Leu Thr Arg Glu 290 295 300 Val Tyr Thr Asp Pro Ile Val Phe Asn Arg Glu Thr Ser Gly Gly Phe 305 310 315 320 Cys Arg Arg Trp Ser Leu Asn Ser Asp Ile Ser Phe Ser Glu Val Glu 325 330 335 Ser Ala Val Ile Arg Ser Pro His Leu Phe Asp Ile Leu Ser Glu Ile 340 345 350 Glu Phe Tyr Thr Thr Arg Ala Gly Leu Pro Leu Asn Asn Thr Glu Tyr 355 360 365 Leu Glu Tyr Trp Val Gly His Ser Ile Lys Tyr Lys Asn Thr Asn Ala 370 375 380 Ser Ser Ala Leu Glu Arg Asn Tyr Gly Thr Ile Thr Ser Asn Lys Ile 385 390 395 400 Lys Tyr Tyr Asp Leu Ala Asn Lys Asp Ile Phe Gln Val Arg Ser Leu 405 410 415 Gly Ala Asp Leu Ala Asn Tyr Tyr Ala Gln Val Tyr Gly Val Pro Tyr 420 425 430 Ala Ser Phe Thr Leu Leu Asp Lys Asn Thr Gly Ser Gly Ser Val Gly 435 440 445 Gly Phe Thr Tyr Ser Lys Pro His Thr Thr Met Gln Val Cys Thr Gln 450 455 460 Asn Tyr Asn Thr Ile Asp Glu Ile Pro Pro Glu Asn Glu Pro Leu Ser 465 470 475 480 Arg Gly Tyr Ser His Arg Leu Ser His Ile Thr Ser Tyr Ser Phe Ser 485 490 495 Lys Asn Ala Ser Ser Pro Ala Arg Tyr Gly Asn Leu Pro Val Phe Ala 500 505 510 Trp Thr His Arg Ser Ala Asp Val Thr Asn Thr Val Tyr Ser Asp Lys 515 520 525 Ile Thr Gln Ile Pro Val Val Lys Ala His Thr Leu Val Ser Gly Thr 530 535 540 Thr Val Ile Lys Gly Pro Gly Phe Thr Gly Gly Asn Ile Leu Lys Arg 545 550 555 560 Thr Ser Ser Gly Pro Leu Ala Tyr Thr Ser Val Ser Val Lys Ser Pro 565 570 575 Leu Ser Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn 580 585 590 Leu Arg Leu Phe Val Thr Ile Ser Gly Thr Arg Ile Tyr Ser Ile Asn 595 600 605 Val Asn Lys Thr Met Asn Lys Gly Asp Asp Leu Thr Phe Asn Thr Phe 610 615 620 Asp Leu Ala Thr Ile Gly Thr Ala Phe Thr Phe Ser Asn Tyr Ser Asp 625 630 635 640 Ser Leu Thr Val Gly Ala Asp Ser Phe Ala Ser Gly Gly Glu Val Tyr 645 650 655 Val Asp Lys Phe Glu Leu Ile Pro Val Asn Ala Thr Phe Glu Ala Glu 660 665 670 Glu Asp Leu Asp Val Ala Lys Lys Ala Val Asn Gly Leu Phe Thr Ser 675 680 685 Lys Lys Asp Ala Leu Gln Thr Ser Val Thr Asp Tyr Gln Val Asn Gln 690 695 700 Ala Ala Asn Leu Val Glu Cys Leu Ser Asp Glu Leu Tyr Pro Asn Glu 705 710 715 720 Lys Arg Met Leu Trp Asp Ala Val Lys Glu Ala Lys Arg Leu Val Gln 725 730 735 Ala Arg Asn Leu Leu Gln Asp Thr Gly Phe Asn Arg Ile Asn Gly Glu 740 745 750 Asn Gly Trp Thr Gly Ser Thr Gly Ile Glu Val Ala Glu Gly Asp Val 755 760 765 Leu Phe Lys Asp Arg Ser Leu Arg Leu Thr Ser Ala Arg Glu Ile Asp 770 775 780 Thr Glu Thr Tyr Pro Thr Tyr Leu Tyr Gln Gln Ile Asp Glu Ser Leu 785 790 795 800 Leu Lys Pro Tyr Thr Arg Tyr Lys Leu Lys Gly Phe Ile Gly Ser Ser 805 810 815 Gln Asp Leu Glu Ile Lys Leu Ile Arg His Arg Ala Asn Gln Ile Val 820 825 830 Lys Asn Val Pro Asp Asn Leu Leu Pro Asp Val Leu Pro Val Asn Ser 835 840 845 Cys Gly Gly Ile Asp Arg Cys Ser Glu Gln Gln Tyr Val Asp Ala Asn 850 855 860 Leu Ala Leu Glu Asn Asn Gly Glu Asn Gly Asn Met Ser Ser Asp Ser 865 870 875 880 His Ala Phe Ser Phe His Ile Asp Thr Gly Glu Ile Asp Leu Asn Glu 885 890 895 Asn Thr Gly Ile Trp Val Val Phe Lys Ile Pro Thr Thr Asn Gly Tyr 900 905 910 Ala Thr Leu Gly Asn Leu Glu Leu Val Glu Glu Gly Pro Leu Ser Gly 915 920 925 Glu Thr Leu Glu Arg Ala Gln Gln Gln Glu Gln Gln Trp Gln Asp Lys 930 935 940 Met Ala Arg Lys Arg Gly Ala Ser Glu Lys Ala Tyr Tyr Ala Ala Lys 945 950 955 960 Gln Ala Ile Asp Arg Leu Phe Ala Asp Tyr Gln Asp Gln Lys Leu Asn 965 970 975 Ser Gly Val Glu Met Ser Asp Met Leu Ala Ala Gln Asn Leu Val Gln 980 985 990 Ser Ile Pro Tyr Val Tyr Asn Asp Ala Leu Pro Glu Ile Pro Gly Met 995 1000 1005 Asn Tyr Thr Ser Phe Thr Glu Leu Thr Asn Arg Leu Gln Gln Ala 1010 1015 1020 Trp Asn Leu Tyr Asp Leu Arg Asn Ala Ile Pro Asn Gly Asp Phe 1025 1030 1035 Arg Asn Gly Leu Ser Asp Trp Asn Ala Thr Ser Asp Val Asn Val 1040 1045 1050 Gln Gln Leu Ser Asp Thr Ser Val Leu Val Ile Pro Asn Trp Asn 1055 1060 1065 Ser Gln Val Ser Gln Gln Phe Thr Val Gln Pro Asn Tyr Arg Tyr 1070 1075 1080 Val Leu Arg Val Thr Ala Arg Lys Glu Gly Val Gly Asp Gly Tyr 1085 1090 1095 Val Ile Ile Arg Asp Gly Ala Asn Gln Thr Glu Thr Leu Thr Phe 1100 1105 1110 Asn Ile Cys Asp Asp Asp Thr Gly Val Leu Ser Ala Asp Gln Thr 1115 1120 1125 Ser Tyr Ile Thr Lys Thr Val Glu Phe Thr Pro Ser Thr Glu Gln 1130 1135 1140 Val Trp Ile Asp Met Ser Glu Thr Glu Gly Val Phe Asn Ile Glu 1145 1150 1155 Ser Val Glu Leu Val Leu Glu Glu Glu 1160 1165 2 3504 DNA Bacillus thuringiensis exon (1)..(3501) 2 atg agt cca aat aat caa aat gaa tat gaa att cta gat gct tca tca 48 Met Ser Pro Asn Asn Gln Asn Glu Tyr Glu Ile Leu Asp Ala Ser Ser 1 5 10 15 tct act tct gta tcc gat aat tct gtt aga tac cct tta gca aac gat 96 Ser Thr Ser Val Ser Asp Asn Ser Val Arg Tyr Pro Leu Ala Asn Asp 20 25 30 caa acg acc aca tta caa aac atg aac tat aaa gat tat ctg aga atg 144 Gln Thr Thr Thr Leu Gln Asn Met Asn Tyr Lys Asp Tyr Leu Arg Met 35 40 45 tct gag gga gag aat cct gaa tta ttt gga aat ccg gag acg ttt att 192 Ser Glu Gly Glu Asn Pro Glu Leu Phe Gly Asn Pro Glu Thr Phe Ile 50 55 60 agt tca tct acg gtt caa act gga att ggc att gtt ggt caa gta ctg 240 Ser Ser Ser Thr Val Gln Thr Gly Ile Gly Ile Val Gly Gln Val Leu 65 70 75 80 ggg gct tta ggg gtt cca ttt gct gga cag ata gct agt ttt tat agt 288 Gly Ala Leu Gly Val Pro Phe Ala Gly Gln Ile Ala Ser Phe Tyr Ser 85 90 95 ttc att gtc ggt caa tta tgg cca tca agt acc gtg agt gta tgg gaa 336 Phe Ile Val Gly Gln Leu Trp Pro Ser Ser Thr Val Ser Val Trp Glu 100 105 110 atg att atg aaa caa gtg gaa gat cta att gat caa aaa ata aca gat 384 Met Ile Met Lys Gln Val Glu Asp Leu Ile Asp Gln Lys Ile Thr Asp 115 120 125 tct gta agg aaa aca gcg ctt gca gga cta caa gga tta gga gat ggc 432 Ser Val Arg Lys Thr Ala Leu Ala Gly Leu Gln Gly Leu Gly Asp Gly 130 135 140 tta gac gta tat cag aaa tca ctt aag aat tgg ctg gaa aat cgt aat 480 Leu Asp Val Tyr Gln Lys Ser Leu Lys Asn Trp Leu Glu Asn Arg Asn 145 150 155 160 gat aca aga gct aga agt gtt gtg gtg acc caa tat ata gct tta gag 528 Asp Thr Arg Ala Arg Ser Val Val Val Thr Gln Tyr Ile Ala Leu Glu 165 170 175 ctt gat ttt gtt gct aaa atc cca tct ttt gca ata tct gga cag gaa 576 Leu Asp Phe Val Ala Lys Ile Pro Ser Phe Ala Ile Ser Gly Gln Glu 180 185 190 gta cca tta tta tca gtg tat gca caa gca gcg aat tta cat ttg cta 624 Val Pro Leu Leu Ser Val Tyr Ala Gln Ala Ala Asn Leu His Leu Leu 195 200 205 tta tta cga gat gct tcc att ttt gga gca gag tgg gga ttc aca cca 672 Leu Leu Arg Asp Ala Ser Ile Phe Gly Ala Glu Trp Gly Phe Thr Pro 210 215 220 gga gaa att tcc aca ttt tat gat cgt cag gtg aca cgt acc gcc caa 720 Gly Glu Ile Ser Thr Phe Tyr Asp Arg Gln Val Thr Arg Thr Ala Gln 225 230 235 240 tac tcg gat tat tgt gta aag tgg tat aac act ggc tta gat aaa tta 768 Tyr Ser Asp Tyr Cys Val Lys Trp Tyr Asn Thr Gly Leu Asp Lys Leu 245 250 255 aaa ggt acg aat gct gca agt tgg ctg aag tat cac caa ttc cga aga 816 Lys Gly Thr Asn Ala Ala Ser Trp Leu Lys Tyr His Gln Phe Arg Arg 260 265 270 gaa atg aca tta ctg gta tta gat tta gta gcg tta ttt cca aac tat 864 Glu Met Thr Leu Leu Val Leu Asp Leu Val Ala Leu Phe Pro Asn Tyr 275 280 285 gac aca cgt acg tat cca atc gaa aca acg gcc caa ctt aca cgg gaa 912 Asp Thr Arg Thr Tyr Pro Ile Glu Thr Thr Ala Gln Leu Thr Arg Glu 290 295 300 gtg tat aca gat cca ata gta ttt aac aga gaa aca agt ggt gga ttt 960 Val Tyr Thr Asp Pro Ile Val Phe Asn Arg Glu Thr Ser Gly Gly Phe 305 310 315 320 tgt agg cgt tgg tca ctt aac agt gat att tct ttt tca gaa gtc gaa 1008 Cys Arg Arg Trp Ser Leu Asn Ser Asp Ile Ser Phe Ser Glu Val Glu 325 330 335 agc gct gta att cgt tca cca cac cta ttt gat ata ctc agt gaa ata 1056 Ser Ala Val Ile Arg Ser Pro His Leu Phe Asp Ile Leu Ser Glu Ile 340 345 350 gaa ttt tat aca aca aga gcg ggg ctt ccc ttg aat aat acg gaa tac 1104 Glu Phe Tyr Thr Thr Arg Ala Gly Leu Pro Leu Asn Asn Thr Glu Tyr 355 360 365 ctt gaa tat tgg gta gga cat tct ata aaa tat aaa aat acg aat gcc 1152 Leu Glu Tyr Trp Val Gly His Ser Ile Lys Tyr Lys Asn Thr Asn Ala 370 375 380 tca tca gca tta gaa cgt aat tac ggt acg att act tct aac aaa atc 1200 Ser Ser Ala Leu Glu Arg Asn Tyr Gly Thr Ile Thr Ser Asn Lys Ile 385 390 395 400 aag tat tat gat tta gca aat aag gat atc ttt cag gtt cga tca tta 1248 Lys Tyr Tyr Asp Leu Ala Asn Lys Asp Ile Phe Gln Val Arg Ser Leu 405 410 415 ggg gcg gat tta gct aat tac tac gca cag gta tat gga gtt ccg tac 1296 Gly Ala Asp Leu Ala Asn Tyr Tyr Ala Gln Val Tyr Gly Val Pro Tyr 420 425 430 gct agt ttt aca ctg ctt gac aag aat aca gga tca gga tca gtt gga 1344 Ala Ser Phe Thr Leu Leu Asp Lys Asn Thr Gly Ser Gly Ser Val Gly 435 440 445 ggt ttt acg tac tca aaa cca cat aca act atg caa gta tgt aca caa 1392 Gly Phe Thr Tyr Ser Lys Pro His Thr Thr Met Gln Val Cys Thr Gln 450 455 460 aat tac aat acg att gat gaa atc cct cca gag aat gag cca ctt agt 1440 Asn Tyr Asn Thr Ile Asp Glu Ile Pro Pro Glu Asn Glu Pro Leu Ser 465 470 475 480 aga ggg tat agc cat aga tta tct cat atc acc tct tat tct ttt tct 1488 Arg Gly Tyr Ser His Arg Leu Ser His Ile Thr Ser Tyr Ser Phe Ser 485 490 495 aag aat gct agt agt cct gct aga tat ggc aat ctc cct gta ttt gct 1536 Lys Asn Ala Ser Ser Pro Ala Arg Tyr Gly Asn Leu Pro Val Phe Ala 500 505 510 tgg aca cat cgg agt gcg gat gtt aca aat aca gtt tat tca gat aaa 1584 Trp Thr His Arg Ser Ala Asp Val Thr Asn Thr Val Tyr Ser Asp Lys 515 520 525 att act cag ata cca gtt gta aag gca cat act tta gtt tca ggt act 1632 Ile Thr Gln Ile Pro Val Val Lys Ala His Thr Leu Val Ser Gly Thr 530 535 540 act gtt att aaa ggt cct gga ttt aca gga ggc aat atc ctt aaa aga 1680 Thr Val Ile Lys Gly Pro Gly Phe Thr Gly Gly Asn Ile Leu Lys Arg 545 550 555 560 aca agt agt ggt ccg tta gct tat act agt gtc tct gta aaa tca cca 1728 Thr Ser Ser Gly Pro Leu Ala Tyr Thr Ser Val Ser Val Lys Ser Pro 565 570 575 tta tca caa aga tat cgt gca aga ata cgt tat gct tct act act aac 1776 Leu Ser Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn 580 585 590 tta cga ctt ttt gta aca att tct gga act cgc att tac tct ata aat 1824 Leu Arg Leu Phe Val Thr Ile Ser Gly Thr Arg Ile Tyr Ser Ile Asn 595 600 605 gtt aat aaa acc atg aat aaa ggg gat gat tta aca ttt aat aca ttt 1872 Val Asn Lys Thr Met Asn Lys Gly Asp Asp Leu Thr Phe Asn Thr Phe 610 615 620 gac tta gca act att ggt act gct ttc aca ttt tca aat tac tcg gat 1920 Asp Leu Ala Thr Ile Gly Thr Ala Phe Thr Phe Ser Asn Tyr Ser Asp 625 630 635 640 agc tta acg gta ggt gca gat tct ttt gct tca gga gga gaa gtt tat 1968 Ser Leu Thr Val Gly Ala Asp Ser Phe Ala Ser Gly Gly Glu Val Tyr 645 650 655 gta gat aag ttc gaa ctt att ccg gta aat gca aca ttt gaa gca gaa 2016 Val Asp Lys Phe Glu Leu Ile Pro Val Asn Ala Thr Phe Glu Ala Glu 660 665 670 gaa gac cta gat gtg gca aag aaa gca gta aat ggc ttg ttt acg agt 2064 Glu Asp Leu Asp Val Ala Lys Lys Ala Val Asn Gly Leu Phe Thr Ser 675 680 685 aaa aaa gat gcc tta cag aca agt gta acg gat tat caa gtg aat caa 2112 Lys Lys Asp Ala Leu Gln Thr Ser Val Thr Asp Tyr Gln Val Asn Gln 690 695 700 gcg gca aac tta gta gaa tgc cta tcc gat gag tta tac cca aat gaa 2160 Ala Ala Asn Leu Val Glu Cys Leu Ser Asp Glu Leu Tyr Pro Asn Glu 705 710 715 720 aaa cga atg tta tgg gat gca gtg aaa gag gcg aaa cga ctt gtt cag 2208 Lys Arg Met Leu Trp Asp Ala Val Lys Glu Ala Lys Arg Leu Val Gln 725 730 735 gca cgt aac tta ctc caa gat aca ggc ttt aat agg att aat gga gaa 2256 Ala Arg Asn Leu Leu Gln Asp Thr Gly Phe Asn Arg Ile Asn Gly Glu 740 745 750 aac gga tgg acg gga agt acg gga atc gag gtt gcg gaa gga gat gtt 2304 Asn Gly Trp Thr Gly Ser Thr Gly Ile Glu Val Ala Glu Gly Asp Val 755 760 765 ctg ttt aaa gat cgt tcg ctt cgt ttg aca agt gcg aga gag att gat 2352 Leu Phe Lys Asp Arg Ser Leu Arg Leu Thr Ser Ala Arg Glu Ile Asp 770 775 780 aca gaa aca tat cca acg tat ctc tat caa caa ata gat gaa tca ctt 2400 Thr Glu Thr Tyr Pro Thr Tyr Leu Tyr Gln Gln Ile Asp Glu Ser Leu 785 790 795 800 tta aaa cca tat aca aga tat aaa cta aaa ggt ttt ata gga agt agt 2448 Leu Lys Pro Tyr Thr Arg Tyr Lys Leu Lys Gly Phe Ile Gly Ser Ser 805 810 815 caa gat tta gag att aaa tta ata cgt cat cgg gca aat caa atc gtc 2496 Gln Asp Leu Glu Ile Lys Leu Ile Arg His Arg Ala Asn Gln Ile Val 820 825 830 aaa aat gta cca gat aat ctc ttg cca gat gta ctc cct gtc aat tct 2544 Lys Asn Val Pro Asp Asn Leu Leu Pro Asp Val Leu Pro Val Asn Ser 835 840 845 tgt ggt ggg atc gat cgc tgc agt gag caa cag tat gta gac gcg aat 2592 Cys Gly Gly Ile Asp Arg Cys Ser Glu Gln Gln Tyr Val Asp Ala Asn 850 855 860 tta gca ctc gaa aac aat gga gaa aat gga aat atg tct tct gat tcc 2640 Leu Ala Leu Glu Asn Asn Gly Glu Asn Gly Asn Met Ser Ser Asp Ser 865 870 875 880 cat gca ttt tct ttc cat att gat aca ggt gaa ata gat ttg aat gaa 2688 His Ala Phe Ser Phe His Ile Asp Thr Gly Glu Ile Asp Leu Asn Glu 885 890 895 aat aca gga att tgg gtc gta ttt aaa att ccg aca aca aat gga tac 2736 Asn Thr Gly Ile Trp Val Val Phe Lys Ile Pro Thr Thr Asn Gly Tyr 900 905 910 gca aca cta gga aat ctt gaa ttg gta gaa gag ggg cca ttg tca ggg 2784 Ala Thr Leu Gly Asn Leu Glu Leu Val Glu Glu Gly Pro Leu Ser Gly 915 920 925 gaa aca tta gaa cga gca caa caa caa gaa caa caa tgg caa gac aaa 2832 Glu Thr Leu Glu Arg Ala Gln Gln Gln Glu Gln Gln Trp Gln Asp Lys 930 935 940 atg gca aga aaa cgt ggg gca tca gaa aaa gca tat tat gca gca aag 2880 Met Ala Arg Lys Arg Gly Ala Ser Glu Lys Ala Tyr Tyr Ala Ala Lys 945 950 955 960 caa gcc att gat cgt tta ttc gca gat tat caa gac caa aaa ctt aat 2928 Gln Ala Ile Asp Arg Leu Phe Ala Asp Tyr Gln Asp Gln Lys Leu Asn 965 970 975 tct ggt gta gaa atg tca gat atg ttg gca gcc caa aac ctt gta cag 2976 Ser Gly Val Glu Met Ser Asp Met Leu Ala Ala Gln Asn Leu Val Gln 980 985 990 tcc att cct tac gta tat aat gat gcg tta cca gaa atc cct gga atg 3024 Ser Ile Pro Tyr Val Tyr Asn Asp Ala Leu Pro Glu Ile Pro Gly Met 995 1000 1005 aac tat acg agt ttt aca gag tta aca aat aga ctc caa caa gca 3069 Asn Tyr Thr Ser Phe Thr Glu Leu Thr Asn Arg Leu Gln Gln Ala 1010 1015 1020 tgg aat ttg tat gat ctt cga aat gct ata cca aat gga gat ttt 3114 Trp Asn Leu Tyr Asp Leu Arg Asn Ala Ile Pro Asn Gly Asp Phe 1025 1030 1035 cga aat gga tta agt gat tgg aat gca aca tca gat gtg aat gtg 3159 Arg Asn Gly Leu Ser Asp Trp Asn Ala Thr Ser Asp Val Asn Val 1040 1045 1050 caa caa cta agc gat aca tct gtc ctt gtc att cca aac tgg aat 3204 Gln Gln Leu Ser Asp Thr Ser Val Leu Val Ile Pro Asn Trp Asn 1055 1060 1065 tct caa gtg tca caa caa ttt aca gtt caa ccg aat tat aga tat 3249 Ser Gln Val Ser Gln Gln Phe Thr Val Gln Pro Asn Tyr Arg Tyr 1070 1075 1080 gtg tta cgt gtc aca gcg aga aaa gag gga gta gga gac gga tat 3294 Val Leu Arg Val Thr Ala Arg Lys Glu Gly Val Gly Asp Gly Tyr 1085 1090 1095 gtg atc atc cgt gat ggt gcg aat cag aca gaa aca ctc aca ttt 3339 Val Ile Ile Arg Asp Gly Ala Asn Gln Thr Glu Thr Leu Thr Phe 1100 1105 1110 aat ata tgt gat gat gat aca ggt gtt tta tct gct gat caa act 3384 Asn Ile Cys Asp Asp Asp Thr Gly Val Leu Ser Ala Asp Gln Thr 1115 1120 1125 agc tat atc aca aaa aca gtg gaa ttc act cca tct aca gag caa 3429 Ser Tyr Ile Thr Lys Thr Val Glu Phe Thr Pro Ser Thr Glu Gln 1130 1135 1140 gtt tgg att gac atg agt gag acc gaa ggt gta ttc aac ata gaa 3474 Val Trp Ile Asp Met Ser Glu Thr Glu Gly Val Phe Asn Ile Glu 1145 1150 1155 agt gta gaa ctc gtg tta gaa gaa gag taa 3504 Ser Val Glu Leu Val Leu Glu Glu Glu 1160 1165 3 3690 DNA Bacillus thuringiensis 3 gaattctaat gacacagtag aatattttta aaataaagat ggaagggggg atatgaaaaa 60 tataatcaca agagtcatac aaaaagatgg ttatgttaaa acaaaaaaat cctgtaggaa 120 taagggttta aaagcaatcg tttgaaaaga tagttatatt aaattgtatg tataggggga 180 aaaaagatga gtccaaataa tcaaaatgaa tatgaaattc tagatgcttc atcatctact 240 tctgtatccg ataattctgt tagataccct ttagcaaacg atcaaacgac cacattacaa 300 aacatgaact ataaagatta tctgagaatg tctgagggag agaatcctga attatttgga 360 aatccggaga cgtttattag ttcatctacg gttcaaactg gaattggcat tgttggtcaa 420 gtactggggg ctttaggggt tccatttgct ggacagatag ctagttttta tagtttcatt 480 gtcggtcaat tatggccatc aagtaccgtg agtgtatggg aaatgattat gaaacaagtg 540 gaagatctaa ttgatcaaaa aataacagat tctgtaagga aaacagcgct tgcaggacta 600 caaggattag gagatggctt agacgtatat cagaaatcac ttaagaattg gctggaaaat 660 cgtaatgata caagagctag aagtgttgtg gtgacccaat atatagcttt agagcttgat 720 tttgttgcta aaatcccatc ttttgcaata tctggacagg aagtaccatt attatcagtg 780 tatgcacaag cagcgaattt acatttgcta ttattacgag atgcttccat ttttggagca 840 gagtggggat tcacaccagg agaaatttcc acattttatg atcgtcaggt gacacgtacc 900 gcccaatact cggattattg tgtaaagtgg tataacactg gcttagataa attaaaaggt 960 acgaatgctg caagttggct gaagtatcac caattccgaa gagaaatgac attactggta 1020 ttagatttag tagcgttatt tccaaactat gacacacgta cgtatccaat cgaaacaacg 1080 gcccaactta cacgggaagt gtatacagat ccaatagtat ttaacagaga aacaagtggt 1140 ggattttgta ggcgttggtc acttaacagt gatatttctt tttcagaagt cgaaagcgct 1200 gtaattcgtt caccacacct atttgatata ctcagtgaaa tagaatttta tacaacaaga 1260 gcggggcttc ccttgaataa tacggaatac cttgaatatt gggtaggaca ttctataaaa 1320 tataaaaata cgaatgcctc atcagcatta gaacgtaatt acggtacgat tacttctaac 1380 aaaatcaagt attatgattt agcaaataag gatatctttc aggttcgatc attaggggcg 1440 gatttagcta attactacgc acaggtatat ggagttccgt acgctagttt tacactgctt 1500 gacaagaata caggatcagg atcagttgga ggttttacgt actcaaaacc acatacaact 1560 atgcaagtat gtacacaaaa ttacaatacg attgatgaaa tccctccaga gaatgagcca 1620 cttagtagag ggtatagcca tagattatct catatcacct cttattcttt ttctaagaat 1680 gctagtagtc ctgctagata tggcaatctc cctgtatttg cttggacaca tcggagtgcg 1740 gatgttacaa atacagttta ttcagataaa attactcaga taccagttgt aaaggcacat 1800 actttagttt caggtactac tgttattaaa ggtcctggat ttacaggagg caatatcctt 1860 aaaagaacaa gtagtggtcc gttagcttat actagtgtct ctgtaaaatc accattatca 1920 caaagatatc gtgcaagaat acgttatgct tctactacta acttacgact ttttgtaaca 1980 atttctggaa ctcgcattta ctctataaat gttaataaaa ccatgaataa aggggatgat 2040 ttaacattta atacatttga cttagcaact attggtactg ctttcacatt ttcaaattac 2100 tcggatagct taacggtagg tgcagattct tttgcttcag gaggagaagt ttatgtagat 2160 aagttcgaac ttattccggt aaatgcaaca tttgaagcag aagaagacct agatgtggca 2220 aagaaagcag taaatggctt gtttacgagt aaaaaagatg ccttacagac aagtgtaacg 2280 gattatcaag tgaatcaagc ggcaaactta gtagaatgcc tatccgatga gttataccca 2340 aatgaaaaac gaatgttatg ggatgcagtg aaagaggcga aacgacttgt tcaggcacgt 2400 aacttactcc aagatacagg ctttaatagg attaatggag aaaacggatg gacgggaagt 2460 acgggaatcg aggttgcgga aggagatgtt ctgtttaaag atcgttcgct tcgtttgaca 2520 agtgcgagag agattgatac agaaacatat ccaacgtatc tctatcaaca aatagatgaa 2580 tcacttttaa aaccatatac aagatataaa ctaaaaggtt ttataggaag tagtcaagat 2640 ttagagatta aattaatacg tcatcgggca aatcaaatcg tcaaaaatgt accagataat 2700 ctcttgccag atgtactccc tgtcaattct tgtggtggga tcgatcgctg cagtgagcaa 2760 cagtatgtag acgcgaattt agcactcgaa aacaatggag aaaatggaaa tatgtcttct 2820 gattcccatg cattttcttt ccatattgat acaggtgaaa tagatttgaa tgaaaataca 2880 ggaatttggg tcgtatttaa aattccgaca acaaatggat acgcaacact aggaaatctt 2940 gaattggtag aagaggggcc attgtcaggg gaaacattag aacgagcaca acaacaagaa 3000 caacaatggc aagacaaaat ggcaagaaaa cgtggggcat cagaaaaagc atattatgca 3060 gcaaagcaag ccattgatcg tttattcgca gattatcaag accaaaaact taattctggt 3120 gtagaaatgt cagatatgtt ggcagcccaa aaccttgtac agtccattcc ttacgtatat 3180 aatgatgcgt taccagaaat ccctggaatg aactatacga gttttacaga gttaacaaat 3240 agactccaac aagcatggaa tttgtatgat cttcgaaatg ctataccaaa tggagatttt 3300 cgaaatggat taagtgattg gaatgcaaca tcagatgtga atgtgcaaca actaagcgat 3360 acatctgtcc ttgtcattcc aaactggaat tctcaagtgt cacaacaatt tacagttcaa 3420 ccgaattata gatatgtgtt acgtgtcaca gcgagaaaag agggagtagg agacggatat 3480 gtgatcatcc gtgatggtgc gaatcagaca gaaacactca catttaatat atgtgatgat 3540 gatacaggtg ttttatctgc tgatcaaact agctatatca caaaaacagt ggaattcact 3600 ccatctacag agcaagtttg gattgacatg agtgagaccg aaggtgtatt caacatagaa 3660 agtgtagaac tcgtgttaga agaagagtaa 3690 

1. A protein having an amino acid sequence described in SEQ. ID. No. 1 in the Sequence Listing and exhibiting a pesticidal activity.
 2. A protein having an amino acid sequence derived by addition, deletion or substitution of a plurality of amino acids in the amino acid sequence described in SEQ. ID. No. 1 in the Sequence Listing and exhibiting a pesticidal activity.
 3. A DNA containing a nucleotide sequence encoding the protein as claimed in claim
 1. 4. The DNA as claimed in claim 3, containing the nucleotide sequence as described in SEQ. ID. No. 3 in the Sequence Listing.
 5. A DNA containing a nucleotide sequence encoding the protein as claimed in claim
 2. 6. A noxious organism-controlling agent, comprising a microbe producing a protein having an amino acid sequence described in SEQ. ID. No. 1 in the Sequence Listing, selected from (1-1) Bacillus thuringiensis serovar galleriae SDS502 strain, (1-2) a mutant thereof, and (1-3) a microbe transformed with a DNA containing a nucleotide sequence encoding a protein having an amino acid sequence described in SEQ. ID. No. 1 in the Sequence Listing, or a protein having a pesticidal activity, produced by a microbe selected from (2-1) the above-mentioned SDS502 strain, (2-2) its mutant, and (2-3) transformed microbe.
 7. A microbe transformed with the DNA as claimed in claim 5 and producing a protein exhibiting the pesticidal activity as claimed in claim
 2. 8. A plant transformed with the DNA as claimed in claim 3 or 5, or a seed thereof
 9. A method for controlling a noxious organism, wherein the protein as claimed in claim 1 or 2 above is fed to a noxious organism to protect a plant from a damage caused by the noxious organism.
 10. The method for controlling a noxious organism as claimed in claim 9, wherein the noxious organism is a Coleoptera insect and the plant is protected from a damage caused by the noxious organism.
 11. Bacillus thuringiensis serovar galleriae SDS502 strain producing a protein having an amino acid sequence described in SEQ. ID. No. 1 in the Sequence Listing and exhibiting a pesticidal activity. 